1. Field of the Invention
The present invention relates to the field of immunochromatographic assays. More particularly, the present invention relates to a method for detecting enteric pathogens in fecal specimens. More particularly yet, the present invention relates to a method for detecting the presence of one or more specific enteric pathogens from the simultaneous assay for several enteric pathogens. Most particularly, the present invention relates to a method for detecting the presence or absence of several enteric pathogens and concurrently applying one or more general tests for an inflammatory condition of the intestines.
2. Description of Prior Art
Enteric pathogens can cause severe illness in people and affect a large number of people within a short period of time. The U.S. Centers for Disease Control estimates that there are five million cases of foodborne illnesses per year in the United States with up to 5,000 deaths. Campylobacter jejuni and Salmonella are the leading causes of foodborne illnesses; E. coli O157 is less frequent, but is significant in disease control because this strain of E. coli, the most noted of the enterohemorrhagic E. coli bacteria, causes the majority of severe disease from E. coli and is also a cause of large epidemics. Other strains of E. coli, such as O111, have also been implicated in foodborne outbreaks. Although the number of illnesses from E. coli O157 is low relative to the numbers of cases of Campylobacter jejuni or Salmonella, this E. coli pathogen is significant because the rate of mortality is much higher and the treatment significantly different. Some antibiotics can have a detrimental effect on patient health if the patient is suffering from an illness caused by an enterohemorrhagic E. coli that has not been diagnosed. For this reason, before prescribing antibiotics for a patient apparently suffering from an illness caused by a pathogen, it is important to determine whether E. coli bacteria is the cause in order to avoid prescribing medication detrimental to the health of the patient. Rapid determination of the cause of the illness is thus a critical factor in providing the timely and proper care of the ill. Moreover, it is critical to determine the possible sources of contamination as quickly as possible in order to take appropriate steps to eliminate them.
A method to rapidly screen patients for enteric illnesses would greatly increase the ability of medical personnel to accurately diagnose the cause of the illness and provide appropriate treatment, thus accelerating the rate of patient recovery. Furthermore, a method for rapid screening for enteric pathogens also has great economic value because it can focus valuable resources. For example, rapid screening can be used to rule out certain pathogens as a cause of illness before making the decision to use the more costly methods of culturing a specimen for a pathogen that cannot be readily detected by a rapid screening device. Use of a method for rapidly screening patients for enteric illnesses that can be performed reliably by unskilled persons is also a more cost-effective use of personnel resources than the use of a method that requires the attention of skilled personnel.
Enteric pathogens are not the only cause of diarrhea. Other causes include food sensitivities, allergic reactions, side effects from medication, and psychosomatic factors. The treatment for diarrhea resulting from such non-pathogenic factors is very different than the anti-microbial treatment of pathogenic diarrhea. A screening method that would give an indication of whether the cause of the diarrhea is infectious or non-infectious, inflammatory or non-inflammatory would provide valuable diagnostic information to a treating physician. A common response to enteric pathogens is an inflammation of the intestines. In contrast, an inflammatory condition of the intestines is generally lacking when the cause of the diarrhea is non-infectious or non-immune. Thus, it is very useful to assay for a marker that is associated with intestinal inflammation and that is absent or present only in very low concentrations in the absence of such inflammation. The detection of such a general marker is useful, for example, in a situation in which none of the specific pathogens assayed for appears to be present. The knowledge that the xe2x80x9cinflammationxe2x80x9d marker is present then will lead to a continued search for the pathogen causing the inflammation, and will also provide additional causes for symptoms such as inflammatory bowel disease. Thus, a method that not only tests for multiple enteric pathogens simultaneously, but also tests for an inflammatory condition of the intestines would provide the physician with additional valuable diagnostic information. Furthermore, a method that would also test for a bacterial, viral, or protozoan cause of enteric disease would provide further valuable diagnostic information in the situation where none of the specific pathogens being assayed for appears to be present.
The immunoassay technique, which relies on the specific binding action between an antigen or a hapten and a corresponding antibody, has proven to be a reliable method for determining the presence (or absence) of a pathogen in a specimen. A class of devices known as immunochromatographic test (ICT) devices uses the immunoassay technique in combination with a label that is conjugated with the antibody and is now commonly used for rapid, reliable field tests to determine the presence or absence of a particular analyte. The label, when attached to antibody/antigen molecules that are then amassed together in a specific, restricted area, becomes readily detectable by the naked human eye, or by a scanning device, depending on the type of label used. In general, the label can be a particle of latex, gold, or carbon, a radioactive particle, a magnetic particle, or have other physical or chemical properties that allow it to be fixed or attracted to a certain defined area. ICT devices that use the sandwich technique are particularly easy to use. With this technique, labeled antibody that binds with the specific antigen to be assayed is mixed with the sample that is suspected of containing the specific antigen. If the antigen is present in the sample, the labeled antibody binds with the antigen to form a label-antibody-antigen complex. A second antibody that is immovably fixed at a test zone and that also binds with the specific antigen binds the label-antibody-antigen complex at the test zone. A positive result is made visible by the accumulation of the label at the test zone. Such devices are economical and can be used by unskilled workers. Thus, a method that uses such an ICT device to determine, in a single assay, the presence or absence of multiple enteric pathogens, in particular, multiple enteric pathogens, plus a general marker for an inflammatory condition of the intestines would provide valuable diagnostic information to a treating physician.
Several types of such ICT devices are known. Most are the xe2x80x9cdipstickxe2x80x9d type in which a test strip is encased in a hollow housing with a bibulous pad extending from one end. This pad is dipped into the liquid sample and draws the liquid by capillary (xe2x80x9cwickingxe2x80x9d) action up onto a section of the test strip that contains a labeled antibody, i.e., a label conjugated to an antibody that will specifically bind with the antigen being assayed. The labeled antibody moves with the liquid that is being drawn by the capillary action further along the test strip and, if the specific antigen to which the antibody binds is present in the liquid sample, the labeled antibody will bind with the antigen, forming a labeled antibody-antigen complex. This complex continues to flow with the liquid along the test strip. Downstream from the area containing the conjugated antibody is a test zone. This test zone is typically a nitrocellulose pad into which a second binding partner, an antibody that binds to the same antigen as the labeled antibody, but to a second epitope of the antigen, has been immovably fixed. The fixed antibody will attach to the labeled antibody-antigen complex that flows onto the test zone and will bind the complex to the test zone. The presence of an antigen being assayed is then visible as a stripe across the test zone or otherwise readily detectable. The excess liquid continues to flow past the test zone across a control zone. There are a number of well-known means in the field of immunoassay of creating a control zone, such as embedding into the control zone a binding partner that binds non-specifically to one or more of the labeled antibodies contained on the conjugate section, or to a labeled analyte added to the liquid sample for the purpose of binding with the non-specific binding partner at the control zone. A properly completed test will always show a visible stripe across the control zone or, if a radioisotope or magnetic particle is used as a label, an otherwise readily detectable stripe. Typically, for those devices using a colored label, the housing of the ICT device has a window through which to view the test zone and the control zone. Devices of this type have been disclosed in May et al. (U.S. Pat. No. 5,622,871; issued Apr. 22, 1997) and Charlton et al. (U.S. Pat. No. 5,714,389; issued Feb. 3, 1998).
Chandler (U.S. Pat. No. 5,869,345; issued 1999, and U.S. Pat. No. 5,877,028; issued 1999) discloses an ICT device that is a two-panel card containing a test strip on one panel and a sample well on the other panel. These patents, which are assigned to SmithKline Diagnostics, Inc. but licensed exclusively to the Assignee of the present invention, are incorporated herein by reference. The Chandler devices also uses the sandwich technique described above with the dipstick devices, but have a particular advantage in that they allow the sample to be prepared for the test directly on the test card, rather than in a separate vessel. When the test card is closed, liquid from the sample well flows onto the test strip. As with the other devices, a window is provided through which to view the results.
Chandler discloses methods for detecting an analyte using the ICT card disclosed in the patents cited above. Similarly, Charlton et al. and May et al. disclose methods for detecting an analyte using the device disclosed in the respective patent. Guerrant et al. (U.S. Pat. No. 5,124,252; issued 1992) discloses a method for distinguishing inflammatory diarrhea from non-inflammatory diarrhea, using radial immunodiffusion assay, latex agglutination assay, or enzyme-linked immunosorbent assay. None of these methods discloses a method for rapid, inexpensive screening for enteric disease that is desirable in diagnosing and treating inflammatory intestinal illness caused by specific enteric pathogens.
What is needed, therefore, is a method that will test for multiple enteric pathogens simultaneously using an ICT device. What is further needed is such a method that will test simultaneously for multiple enteric foodborne pathogens. What is yet further needed is such a method that will indicate an inflammatory condition of the intestines. What is still further needed is such a method that will indicate a bacterial, viral, or protozoan cause of enteric disease. Finally, what is needed is such a method that will provide rapid screening ability, is economical, and can be reliably used by unskilled persons.
It is an object of the present invention to provide a method that will test simultaneously for multiple enteric pathogens. It is a further object of the present invention to provide such a method that will test for multiple enteric foodborne pathogens. It is a yet further object to provide such a method that will test for an inflammatory condition of the intestines. It is a still further object to provide such a method that will test for a bacterial, viral, or protozoan cause of enteric disease.
Finally it is an object of the present invention to provide such a method that will provide highly accurate results within a few minutes, is economical, and can be reliably used by unskilled persons.
The present invention is a method for testing a fecal sample for the presence of one or more enteric pathogens, such a E. coli O157, Campylobacter jejuni, Salmonella, Listeria, Shigella, and Yersinia, Cryptosporidium, Giardia, or a subcombination thereof. These pathogens are common causes of enteric illness or are significant because of a high rate of mortality. It should be understood, however, that the method of the present invention can be used to screen for any number of enteric pathogens including such enteric pathogens as E. coli O26, E. coli O103, E. coli O111, E. coli O121, and E. coli O145. The method of the present invention also tests for the presence of fecal lactoferrin as an indicator of inflammation, and for a bacterial, viral, or protozoan cause of inflammation. The method tests for antigens of the enteric pathogens and the lactoferrin, and for bacterial, viral, or protozoan cause of inflammation, on a single test strip of an ICT device. The method uses the sandwich technique of binding a labeled antibody to a specific antigen and fixing the labeled antigen at a test zone by means of an immovably fixed second antibody that also binds to the specific antigen. The terms xe2x80x9cantigenxe2x80x9d and xe2x80x9cantibodyxe2x80x9d are used herein in the singular form to refer to a plurality of exemplars of the same particular antigen or antibody, respectively.
According to the method of the present invention, liquid from a fecal sample is applied to an immunochromatographic test device having a detection area and a carrier bearing multiple specific labeled antibodies for the multiple antigens being assayed for, each specific antibody binding to one specific antigen of the multiple antigens. As mentioned above, numerous types of such devices are known. Generally, the labeled antibodies are mixed with the liquid from the sample and this mixture then flows onto the detection area. In some devices, the liquid from the sample may first flow onto the detection area and the labeled antibodies then be admixed onto the detection area. The description that follows is based on a unidirectional flow device in which the flow of liquid to be tested is such that the labeled antibodies are mixed with the liquid and the mixture then flows onto the detection area and then onto the control area. It should be understood, however, that the sequence in which the labeled antibodies are mixed with the liquid from the sample is not critical to the immunochromatographic test, as long as the device provides for a mixture at the detection area of labeled antibodies and the liquid to be tested. Thus, bidirectional flow devices, such as are disclosed in Chandler, supra, can also be used to perform the test according to the method of the present invention. The labeled antibodies are conjugated to a label, such as a gold, colored latex, or carbon particle, an enzyme, a magnetic particle, a radioisotope, or other type of label that will allow a chromatographic indication of a positive or negative result. The liquid from the sample will cause these labeled antibodies to be released from the carrier and they will then flow with the liquid. If an antigen being assayed for is present in the liquid, the specific antibody will attach to it, forming a label-antibody-antigen complex with the label attached to the antibody serving as a visual label for the antigen. The liquid then flows onto a detection area such as a nitrocellulose pad or other carrier that contains multiple test zones and a control zone. Each test zone is separate and distinct from every other test zone and carries a second specific antibody for one of the antigens being assayed. This second antibody attaches to the same antigen that the labeled antibody attached to, but to a second epitope on the antigen. For the sake of simplicity, we will refer to this second antibody as a complementary antibody. The complementary antibody is immovably fixed on its test zone so that when liquid containing an antibody-antigen-label complex flows across a test zone that bears the complementary antibody specific to the antigen in the complex, the complementary antibody will attach to that antigen and fix the complex at that test zone. No labels are deposited at a test zone for which the specific antigen is not present. The test zones are readable by label-specific means. By that we mean, the human eye can read the test zones when a colored label is used; a radioisotope scanner or magnetic scanner can be used to read the test zones when radioisotopes or magnetic particles are used as the label, respectively.
After flowing past the series of test zones, the liquid flows across a control zone. The control zone contains a non-specific antibody such as goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG that will bind with any labeled antibodies entrained in the liquid. Thus, the control zone will always have a positive stripe if the liquid sample has flowed the entire length of the test strip. A missing control zone stripe indicates that liquid carrying labeled antibodies did not flow across the control zone and, thus, may not have flowed across the test zones.
Using the technique of applying multiple test zones to an ICT test strip provides a rapid, economical, easy-to-use method of screening fecal samples for multiple enteric pathogens. In particular, the method can be used for rapid screening of the most common enteric pathogens that lead to outbreaks of diarrhea and/or for those enteric pathogens that require very specific treatment or have a particularly high mortality rate. Test kits that test according to the method of the present invention can be prepared for different geographical locations, taking into consideration the particularly relevant pathogens of the area. For example, test kits prepared for Australia or other parts of the world can include a test for E. coli O111, a pathogen that is rarely the cause of foodborne illness in the United States but is a significant cause in Australia.
Diarrhea may be caused by factors other than invasive enteric pathogens, and it is particularly useful for the treating physician to know whether, in the absence of various pathogens that are typically assayed, the diarrhea is associated with an inflammatory condition of the intestines or has other, non-inflammatoryxe2x80x94and hence, generally, non-pathogenicxe2x80x94causes. Lactoferrin is an iron-binding bactericidal protein contained in granules in polymorphonuclear (PMN) leukocytes and is found in intestinal secretions, as well as in other secretions. Since the PMNs increase rapidly in number in response to an infection, the number of lactoferrin granules also increases. Studies have shown that the presence of fecal lactoferrin is a reliable general indicator of inflammation in the intestinal tract. See Choi et al.; To Culture or Not to Culture: Fecal Lactoferrin Screening for Inflammatory Bacterial Diarrhea, in: Journal of Clinical Microbiology, April 1996, p. 928-932. Because lactoferrin is also found in breast milk, fecal lactoferrin found in breast-fed infants is not an effective indicator of an inflammatory intestinal condition, as the presence of lactoferrin from breast milk will lead to false positives. Nevertheless, a method for detecting multiple enteric foodborne pathogens that includes a general indicator for an inflammatory condition of the intestines will provide the physician who is treating a patient other than a breast-fed infant with valuable information.
The assay for the presence of fecal lactoferrin according to the method of the present invention is similar to the assays for enteric pathogensxe2x80x94a first antibody that will bind specifically to the lactoferrin is conjugated to a label and a complementary antibody that will bind specifically to another epitope of the lactoferrin is immovably fixed on a lactoferrin test zone. If lactoferrin is present in the fecal sample, it will be labeled and fixed at the lactoferrin test zone and show up as a readable stripe across the test strip.
An assay that would indicate that the inflammatory enteric disease is caused by a bacterial, viral, or protozoan infection would provide further useful information to treating physicians. For example, if the test results for specific pathogens are negative and the test result for fecal lactoferrin is positive, the treating physician would like to know whether treatment with antibiotics is indicated. Antibiotics are effective against bacterial infections, but ineffective in combating viral infections. Furthermore, it may be detrimental to the health of the patient to prescribe antibiotics when not otherwise indicated and it is certainly not cost-effective to prescribe them as treatment for viral infection.
The assays for a bacteria marker, a virus marker, and a protozoa marker are similar to the assays described above for enteric pathogens and fecal lactoferrin. The assays for detecting markers may use a cocktail of antibodies or provide for multiple stripings of various antibodies. To detect a bacteria marker, for example, a cocktail containing various antibodies for common causes of infection may be applied to the bacteria marker test zone, or a series of test zones, each one marking a different bacteria, may be incorporated on an ICT test strip. Whether a cocktail of antibodies or multiple stripings is used, the immunochromatographic assay method remains the same: a first antibody that will bind to a particular first epitope on a bacteria is conjugated to a label and a complementary antibody that will bind specifically to a particular second epitope on the same bacteria is immovably fixed on a bacterial test zone. When a cocktail is used, the particular bacteria causing the infection will not be identified. A positive result for the bacteria marker will merely indicate that the patient is suffering from a bacterial infection caused by one of the bacteria to which antibodies were included in the cocktail. Similarly, assays for virus and protozoa markers can also be included on the test strip.
In the Preferred Embodiment, a two-panel device as disclosed by Chandler is used to test for the multiple pathogens, the lactoferrin, and the markers for bacteria and virus, although any ICT device that uses the sandwich technique for immunochromatographic assay is suitable for the method of the present invention.